Title: Summary of last lesson
1Summary of last lesson
- Excellent review of techniques for pop gen
- Methods of analysis
- Previous lesson density dependence/janzen
connel/red queen hypothesis/type of markers - humnogous fungus
- Testing the marker/testing sample size
2Frequency-, or density dependent, or balancing
selection
- New alleles, if beneficial because linked to a
trait linked to fitness will be positively
selected for. - Example two races of pathogen are present, but
only one resistant host variety, suggests second
pathogen race has arrived recently
3 - Rapid generation time of pathogens. Reticulated
evolution very likely. Pathogens will be selected
for INCREASED virulence - In the short/medium term with long lived trees a
pathogen is likely to increase its virulence - In long term, selection pressure should result in
widespread resistance among the host
4Overview
- Armillaria bulbosa (gallica)
- Known as the Humungous Fungus, or honey mushroom
- Form rhizomorphs, which make up much of the
humungous part - Basidiocarp cap 6 cm in diameter, stem is 5-10
cm tall - Facultative tree root pathogen
5Life cycle Reproduction
- Sexual
- Basidiocarps release spores (n) after karyogamy
and meiosis - 2 mating-type loci, each with multiple alleles in
the population - Isolates (n) must have different alleles at two
mating type loci to be sexually compatible - Asexual
- vegetative spreading of rhizomorph
- The large mass of rhizomorph that is genetically
isolated is called a clone
6Building up the question
- By extending the areas sampled in subsequent
years, we were finally able to delimit the large
area occupied by this genotype and then go on to
show that this genotype likely represents and
individual -
- - Myron Smith
7Researchers Question
- The clonal individual is especially difficult
to define because the network of hyphae is
underground - How do you unambiguously identify an individual
fungi within a local population?
8Approach
- 1. Collect samples
- 2. Check mating type
- - Somatic compatibility test
- - Distrubution of mating-type alleles
- 3. Molecular testing
- - RFLP
- - RAPD
- 4. Statistics
- 5. More testing
9Methods and Materials 1
- 1. Collecting samples
- Researcher collected samples over a 30 hectare
area by baiting Armillaria with poplar stakes and
taking tissues and spores - They then grew the successfully colonized stakes
in soil taken from the study site - Each fungal colony cultured was called an isolate.
10Methods and Materials 2
Example (not Armillaria)
- 2. Checking mating type
- - Somatic incompatibilityFor two fungal
isolates to fuse, all somatic compatibility loci
must be the same. - Fusion means theyre clones ?
11Methods and Materials 2
- 2. Checking mating type
- - Distrubution of mating alleles
- Mating occurs only when coupled isolates have
different alleles at two unlinked, multiallelic
loci A and B. (They have an incompatibility
system) - If fruit bodies had the same alleles at A and B,
and were collected from the same area, they were
assumed to be from the same clone
12Result 1
- Somatic compatilbilty
- isolates from vegetative mycelium from a large
sampling area fused - Mating alleles
- They had the same mating type
13Result 1
- Clone 1 was found to exceed 500 m in diameter
- Used previously collected mtDNA restriction
fragment patterns
14Sensitivity of Approach
- Problem These tests alone are not enough to
distinguish a clone from closely related
individuals
15Why?
- Q The first two tests were not sensitive enough
to tell a clone from a close relativeWhy? - A Spores from same point source have the same
mating-type alleles, but the offspring they
produce after inbreeding are genetically
distinct.
16Methods and Materials 3
- 3. Molecular Testing
-
- - RFLP analysis at 5 polymorphic, heterozyg. loci
of mtDNA from Clone 1 - - RAPD analysis at 11 loci
17RAPDS vs. RFLPs
- Use 1 short PCR primer
- When it finds match on template at a distance
that can be amplified (primer binds twice within
50 to 2000 bp) RAPD amplicon - Dominant, annoymous
- Total genomic, vs single locus
- Use endonuclease to digest DNA at specific
restriction site - Run digest and see how amplicon was cut
- Single locus is co-dominant
18Result 2
- RFLP
- All 5 loci from Clone 1 were heterozygous and
identical (both alleles present at loci
1,1) - RAPD
- All 11 RAPD products were present in all
vegetative isolates
19Statistical Analysis
- The probability of retaining heterozygosity at
each parental locus in an individual produced by
mating of sibling monospore isolates - 0.0013
- So they were pretty confident that cloning was
responsible for their results, not inbreeding
20More testing, just in case
- To be completely confident, they tested
- 1) that nearby Clone 2 was different and lacked 5
of the Clone 1 heterozyg. RAPD fragments, - 2) more loci, totaling
- 20 RAPD fragments
- 27 nuclear DNA RFLP fragments
- all were identical in Clone 1
21Sensitivity of RAPDs
- Tested on subset of spores from same basidiocarp
- RAPDs differentiated among full sibs
22Conclusions
- Somatic compatibility, mating allele loci, mtDNA,
RFLP, and RAPD tests all indicate that a single
organism could indeed occupy a 15 hectare area
23Conclusions
- The larger individual, Clone 1 was estimated to
weigh 9700 kg and be over 1500 years old
24Implications
- ?????
- Fungi are one of the oldest and largest organisms
on the planet - Recycle nutrientsvery important!
- Armillaria bulbosa also a pathogen its effects
on forest above may be huge as well.
25HOST-SPECIFICITY
- Biological species
- Reproductively isolated
- Measurable differential size of structures
- Gene-for-gene defense model
- Sympatric speciation Heterobasidion, Armillaria,
Sphaeropsis, Phellinus, Fusarium forma speciales
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27Phylogenetic relationships within the
Heterobasidion complex
Fir-Spruce
Pine Europe
Pine N.Am.
28The biology of the organism drives an epidemic
- Autoinfection vs. alloinfection
- Primary spreadby spores
- Secondary spreadvegetative, clonal spread, same
genotype . Completely different scales (from
small to gigantic) - Coriolus
- Heterobasidion
- Armillaria
- Phellinus
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30OUR ABILITY TO
- Differentiate among different individuals
(genotypes) - Determine gene flow among different areas
- Determine allelic distribution in an area
31WILL ALLOW US TO DETERMINE
- How often primary infection occurs or is disease
mostly chronic - How far can the pathogen move on its own
- Is the organism reproducing sexually? is the
source of infection local or does it need input
from the outside
32IN ORDER TO UNDERSTAND PATTERNS OF INFECTION
- If John gave directly Mary an infection, and Mary
gave it to Tom, they should all have the same
strain, or GENOTYPE (comparisonsecondary spread
among forest trees) - If the pathogen is airborne and sexually
reproducing, Mary John and Tom will be infected
by different genotypes. But if the source is the
same, the genotypes will be sibs, thus related
33Recognition of self vs. non self
- Intersterility genes maintain species gene pool.
Homogenic system - Mating genes recognition of other to allow for
recombination. Heterogenic system - Somatic compatibility protection of the
individual.
34Recognition of self vs. non self
- What are the chances two different individuals
will have the same set of VC alleles? - Probability calculation (multiply frequency of
each allele) - More powerful the larger the number of loci
- and the larger the number of alleles per locus
35Recognition of self vs. non self
- It is possible to have different genotypes with
the same vc alleles - VC grouping and genotyping is not the same
- It allows for genotyping without genetic tests
- Reasons behing VC system protection of
resources/avoidance of viral contagion
36Somatic incompatibility
37More on somatic compatibility
- Perform calculation on power of approach
- Temporary compatibility allows for cytoplasmic
contact that then is interrupted this temporary
contact may be enough for viral contagion
38SOMATIC COMPATIBILITY
- Fungi are territorial for two reasons
- Selfish
- Do not want to become infected
- If haploids it is a benefit to mate with other,
but then the nn wants to keep all other
genotypes out - Only if all alleles are the same there will be
fusion of hyphae - If most alleles are the same, but not all, fusion
only temporary
39SOMATIC COMPATIBILITY
- SC can be used to identify genotypes
- SC is regulated by multiple loci
- Individual that are compatible (recognize one
another as self, are within the same SC group) - SC group is used as a proxy for genotype, but in
reality, you may have some different genotypes
that by chance fall in the same SC group - Happens often among sibs, but can happen by
chance too among unrelated individuals
40Recognition of self vs. non self
- What are the chances two different individuals
will have the same set of VC alleles? - Probability calculation (multiply frequency of
each allele) - More powerful the larger the number of loci
- and the larger the number of alleles per locus
41Recognition of self vs. non selfprobability of
identity (PID)
- 4 loci
- 3 biallelelic
- 1 penta-allelic
- P 0.5x0.5x0.5x0.20.025
- In humans 99.9, 1000, 1 in one million
42INTERSTERILITY
- If a species has arisen, it must have some
adaptive advantages that should not be watered
down by mixing with other species - Will allow mating to happen only if individuals
recognized as belonging to the same species - Plus alleles at one of 5 loci (S P V1 V2 V3)
43INTERSTERILITY
- Basis for speciation
- These alleles are selected for more strongly in
sympatry - You can have different species in allopatry that
have not been selected for different IS alleles
44MATING
- Two haploids need to fuse to form nn
- Sex needs to increase diversity need different
alleles for mating to occur - Selection for equal representation of many
different mating alleles
45MATING
- If one individuals is source of inoculum, then
the same 2 mating alleles will be found in local
population -
- If inoculum is of broad provenance then multiple
mating alleles should be found
46MATING
- How do you test for mating?
-
- Place two homokaryons in same plate and check for
formation of dikaryon (microscopic clamp
connections at septa)
47Clamp connections
48MATING ALLELES
- All heterokaryons will have two mating allelels,
for instance a, b - There is an advantage in having more mating
alleles (easier mating, higher chances of finding
a mate) - Mating allele that is rare, may be of migrant
just arrived - If a parent is important source, genotypes should
all be of one or two mating types
49Two scenarios
- A, A, B, C, D, D, E, H, I, L
50Two scenarios
- A, A, B, C, D, D, E, H, I, L
- Multiple source of infections (at least 4
genotypes)
- A, A, A,B, B, A, A
- Siblings as source of infection (1 genotype)
51SEX
- Ability to recombine and adapt
- Definition of population and metapopulation
- Different evolutionary model
- Why sex? Clonal reproductive approach can be very
effective among pathogens
52Long branches in between groups suggests no sex
is occurring in between groups
Fir-Spruce
Pine Europe
Pine N.Am.
53Small branches within a clade indicate sexual
reproduction is ongoing within that group of
individuals
NA S
NA P
EU S
890 bp CIgt0.9
EU F
54Index of association
- Ia if same alleles are associated too much as
opposed to random, it means sex is not occurring - Association among alleles calculated and compared
to simulated random distribution
55Evolution and Population genetics
- Positively selected genes
- Negatively selected genes
- Neutral genes normally population genetics
demands loci used are neutral - Loci under balancing selection..
56Evolution and Population genetics
- Positively selected genes
- Negatively selected genes
- Neutral genes normally population genetics
demands loci used are neutral - Loci under balancing selection..
57Evolutionary history
- Darwininan vertical evolutionary models
- Horizontal, reticulated models..
58Phylogenetic relationships within the
Heterobasidion complex
Fir-Spruce
Pine Europe
Pine N.Am.
59Geneaology of S DNA insertion into P ISG
confirms horizontal transfer.Time of
cross-over uncertain
NA S
NA P
EU S
890 bp CIgt0.9
EU F
60Because of complications such as
- Reticulation
- Gene homogeneization(Gene duplication)
- Need to make inferences based on multiple genes
- Multilocus analysis also makes it possible to
differentiate between sex and lack of sex
(Iaindex of association), and to identify
genotypes, and to study gene flow
61Basic definitions again
- Locus
- Allele
- Dominant vs. codominant marker
- RAPDS
- AFLPs
62How to get multiple loci?
- Random genomic markers
- RAPDS
- Total genome RFLPS (mostly dominant)
- AFLPS
- Microsatellites
- SNPs
- Multiple specific loci
- SSCP
- RFLP
- Sequence information
- Watch out for linked alleles (basically you are
looking at the same thing!)
63RAPDS use short primers but not too short
- Need to scan the genome
- Need to be readable
- 10mers do the job (unfortunately annealing
temperature is pretty low and a lot of priming
errors cause variability in data)
64RAPDS use short primers but not too short
- Need to scan the genome
- Need to be readable
- 10mers do the job (unfortunately annealing
temperature is pretty low and a lot of priming
errors cause variability in data)
65RAPDS can also be obtained with Arbitrary Primed
PCR
- Use longer primers
- Use less stringent annealing conditions
- Less variability in results
66Result series of bands that are present or
absent (1/0)
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68Root disease center in true fir caused by H.
annosum
69Ponderosa pine
Incense cedar
70Yosemite Lodge 1975 Root disease centers
outlined
71Yosemite Lodge 1997 Root disease centers
outlined
72WORK ON PINES HAD DEMONSTRATED INFECTIONS ARE
MOSTLY ON STUMPS
- Use meticulous field work and genetics
information to reconstruct disease from infection
to explosion - On firs/sequoia if the stump theory were also
correct we would find a stump within the outline
of each genotype
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79Are my haplotypes sensitive enough?
- To validate power of tool used, one needs to be
able to differentiate among closely related
individual - Generate progeny
- Make sure each meiospore has different haplotype
- Calculate P
80RAPD combination1 2
- 1010101010
- 1010101010
- 1010101010
- 1010101010
- 1010000000
- 1011101010
- 1010111010
- 1010001010
- 1011001010
- 1011110101
81Conclusions
- Only one RAPD combo is sensitive enough to
differentiate 4 half-sibs (in white) - Mendelian inheritance?
- By analysis of all haplotypes it is apparent that
two markers are always cosegregating, one of the
two should be removed
82If we have codominant markers how many do I need
- IDENTITY tests probability calculation based
on allele frequency Multiplication of
frequencies of alleles - 10 alleles at locus 1 P10.1
- 5 alleles at locus 2 P20,2
- Total P P1P20.02
83Have we sampled enough?
- Resampling approaches
- Saturation curves
- A total of 30 polymorphic alleles
- Our sample is either 10 or 20
- Calculate whether each new sample is
characterized by new alleles
84Saturation (rarefaction) curves
No Of New alleles
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
85Dealing with dominant anonymous multilocus markers
- Need to use large numbers (linkage)
- Repeatability
- Graph distribution of distances
- Calculate distance using Jaccards similarity
index
86Jaccards
- Only 1-1 and 1-0 count, 0-0 do not count
- 1010011
- 1001011
- 1001000
87Jaccards
- Only 1-1 and 1-0 count, 0-0 do not count
- A 1010011 AB 0.6 0.4 (1-AB)
- B 1001011 BC0.5 0.5
- C 1001000 AC0.2 0.8
88Now that we have distances.
- Plot their distribution (clonal vs. sexual)
89Now that we have distances.
- Plot their distribution (clonal vs. sexual)
- Analysis
- Similarity (cluster analysis) a variety of
algorithms. Most common are NJ and UPGMA
90Now that we have distances.
- Plot their distribution (clonal vs. sexual)
- Analysis
- Similarity (cluster analysis) a variety of
algorithms. Most common are NJ and UPGMA - AMOVA requires a priori grouping
91AMOVA groupings
- Individual
- Population
- Region
- AMOVA partitions molecular variance amongst a
priori defined groupings
92Example
- SPECIES X 50blue, 50 yellow
93AMOVA example
Scenario 1
Scenario 2
v
POP 1
POP 2
v
94Expectations for fungi
- Sexually reproducing fungi characterized by high
percentage of variance explained by individual
populations - Amount of variance between populations and
regions will depend on ability of organism to
move, availability of host, and - NOTE if genotypes are not sensitive enough so
you are calling the same things that are
different you may get unreliable results like 100
variance within pops, none among pops
95Results Jaccard similarity coefficients
P. nemorosa
P. pseudosyringae U.S. and E.U.
96P. pseudosyringae genetic similarity patterns are
different in U.S. and E.U.
97Results P. nemorosa
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99The scale of disease
- Dispersal gradients dependent on propagule size,
resilience, ability to dessicate, NOTE not
linear - Important interaction with environment, habitat,
and niche availability. Examples Heterobasidion
in Western Alps, Matsutake mushrooms that offer
example of habitat tracking - Scale of dispersal (implicitely correlated to
metapopulation structure)---
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103RAPDSgt not used often now
104RAPD DATA W/O COSEGREGATING MARKERS
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106PCA
107AFLP
- Amplified Fragment Length Polymorphisms
- Dominant marker
- Scans the entire genome like RAPDs
- More reliable because it uses longer PCR primers
less likely to mismatch - Priming sites are a construct of the sequence in
the organism and a piece of synthesized DNA
108How are AFLPs generated?
- AGGTCGCTAAAATTTT (restriction site in red)
- AGGTCG CTAAATTT
- Synthetic DNA piece ligated
- NNNNNNNNNNNNNNCTAAATTTTT
- Created a new PCR priming site
- NNNNNNNNNNNNNNCTAAATTTTT
- Every time two PCR priming sitea are within
400-1600 bp you obtain amplification
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111Distances between study sites
White mangroves Corioloposis caperata
112Forest fragmentation can lead to loss of gene
flow among previously contiguous populations.
The negative repercussions of such genetic
isolation should most severely affect highly
specialized organisms such as some
plant-parasitic fungi.
AFLP study on single spores
Coriolopsis caperata on Laguncularia racemosa
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115Using DNA sequences
- Obtain sequence
- Align sequences, number of parsimony informative
sites - Gap handling
- Picking sequences (order)
- Analyze sequences (similarity/parsimony/exhaustive
/bayesian - Analyze output CI, HI Bootstrap/decay indices
116Using DNA sequences
- Testing alternative trees kashino hasegawa
- Molecular clock
- Outgroup
- Spatial correlation (Mantel)
- Networks and coalescence approaches
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119From Garbelotto and Chapela, Evolution and
biogeography of matsutakes
Biodiversity within species as significant as
between species
120Microsatellites or SSRs
- AGTTTCATGCGTAGGT CG CG CG CG CG
AAAATTTTAGGTAAATTT - Number of CG is variable
- Design primers on FLANKING region, amplify DNA
- Electrophoresis on gel, or capillary
- Size the allele (different by one or more
repeats if number does not match there may be
polimorphisms in flanking region) - Stepwise mutational process (2 to 3 to 4 to 3 to2
repeats)